Method for quantitative measurement of gene expression for indentifying individuals at risk for bronchogenic carcinoma

ABSTRACT

A method measure expression of multiple target genes in a progenitor cell for bronchogenic carcinoma comprising the use of reverse transcription-polymerase chain reaction (RT-PCR) to allow simultaneous expression measurement of the multiple target genes is disclosed.

[0001] The present invention was made under a Research Grant No. NIH-P01ES07168 from the National Institute of Health who may have certainrights thereto. The present invention relates generally to a method forthe quantitative measurement of gene expression using multiplexcompetitive reverse transcription polymerase chain reaction (MC RT-PCR).To identify individuals at risk for bronchogenic carcinoma.

TECHNICAL BACKGROUND

[0002] The PCR techniques are generally described in U.S. Pat. Nos.4,683,195; 4,683,202 and 4,965,188. The PCR technique generally involvesa process for amplifying any desired specific nucleic acid sequencecontained within a nucleic acid molecule. The PCR process includestreating separate complementary strains of the nucleic acid with anexcess of two oligonucleotide primers. The primers are extended to formcomplementary primer extension products which act as templates forsynthesizing the desired nucleic acid sequence. The PCR process iscarried out in a simultaneous step-wise fashion and can be repeated asoften as desired in order to achieve increased levels of amplificationof the desired nucleic acid sequence. According to the PCR process, thesequence of DNA between the primers on the respective DNA strains areamplified selectively over the remaining portions of the DNA andselected sample. The PCR process provides for the specific amplificationof a desired region of DNA.

[0003] The method of the present invention uses the PCR amplificationprocess that allows simultaneous amplification of a “target gene”, a“housekeeping” gene and competitive templates for each of these genes.According to the present invention, the terms “target DNA sequence” and“target gene” generally refer to a gene of interest for which there is adesire to selectively amplify that gene or DNA sequence. The term“housekeeping” gene refers to genes that are suitable as internalstandards for amount of RNA per PCR reaction. In a general and overallsense, a key to the present invention is the simultaneous use of primersfor target genes, primers for a housekeeping gene, and two internalstandard competitive templates comprising mutants of the target genesand housekeeping gene. These mutations can be point mutations,insertions, deletions or the like.

[0004] There is a need for quantitative measurement of gene expressionwhich controls for the expression of all relevant genes that may beinvolved in individuals at risk for certain diseases, including, forexample, bronchogenic carcinoma. The present invention addresses theseneeds by providing a method for gene expression measurement byquantitative RT-PCR that allows simultaneous expression measurement ofmany genes. The multiplex competitive reverse transcriptase-polymerasechain reaction is generally described in the Willey and Willey et al.U.S. Pat. Nos. 5,639,606; 5,643,765 and 5,876,978 which are fullyincorporated herein by reference, along with all other referencesdisclosed herein and listed at the end of the specification. Accordingto one aspect of the present invention, the mRNA expression of mGST,GSTM3, GSTT1, GSTP1, GSHPx and GSHPxA and the combined expression ofGSTM1, 2, 4, 5 are simultaneously measured in the primary NBECs ofnon-lung cancer patients, primary NBECs from lung cancer patients, andin cultured NBECs from non-lung cancer patients.

[0005] Normal bronchial epithelial cells (NBECs) are at an increasedrisk for oxidative damage following inhalational exposure to reactiveoxygen species in cigarette smoke (1, 2), ozone (3), possibly asbestos(4), and other particulates in the environment. NBECs also are exposedto endogenous oxidative products produced through normal cellularmetabolism (5) and during inflammation (6, 7). In addition, inhaleddaughters of radon-2222 decay (polonium-218 and polonium-214) maydeposit on NBECs and emit α particles that generate reactive oxygenproducts as they encounter the cells. NBECs also are exposed throughinhaled cigarette smoke or urban air pollution to polycylic aromatichydrocarbons (PAHs). These procarcinogens may be metabolically activatedin the cytoplasm and subsequently damage nuclear DNA. Damage to NBECsand adjacent structures from oxidants and/or activated carcinogens mayresult in a variety of pulmonary disorders, including bronchogeniccarcinoma, pulmonary fibrosis, chronic bronchitis, and emphysema (5, 8).

[0006] NBECs express several enzymes, includingglutathione-S-transferase (GSTs) and glutathione peroxidases, that arecapable of preventing or reducing injury from reactive oxidants orcarcinogens. The GST enzymes conjugate reactive chemical groups,including reactive oxygen species and diol-epoxide ultimate carcinogens,to glutathione and thereby prevent them from binding to and damaging DNA(9). There are several classes of GSTs, including one microsomal class(mGST) and four cytosolic classes: GSTA, GSTM, GSTP, and GSTT (10, 11).In addition, a human homologue of rat GSTK1 has been reported (12). EachGST enzyme has substrate specificity, but there is considerable overlap(13). For example, diol-epoxides derive from PAH procarcinogens aremetabolized by GSTP1 and GSTM1-3 (14). Other substrates for thecytosolic GSTs include steroids, alkenals, and quinones (9). In contrastto the cytosolic GST enzymes, mGST has very little specificity forepoxides (15). However, mGST has activity against a broad range of othersubstrates, including styrene-7-8-oxide (16),1-chloro-2,4-dinitrobenzene, and cumene hydroperoxide (17). Further,various halogenated alkynes and alkenes are metabolized preferentiallyby mGST compared to the cytosolic forms (13, 18).

[0007] The glutathione peroxidase enzymes catalyze the inactivation ofperoxides (including hydrogen peroxide and lipid peroxides) usingreduced glutathione as a cofactor (19). Several enzymes have glutathioneperoxidase activity, including GSHPx (19), GSHPxA (a secreted form; Ref.20), mGST (21), GSTA (22), and GSTM3 (23).

[0008] Both intertissue and interindividual variation in the expressionof GST and glutathione peroxidase genes have been reported (14, 24-27).In addition, the expression of some GST and glutathione peroxidase genesis altered in carcinoma tissues (14, 20, 24, 25, 28, 29). Because thereis intertissue variation in the expression of these genes, it isimportant to measure expression specifically in the progenitor cell forbronchogenic carcinoma, the bronchial epithelial cell. There is verylittle information presently available regarding quantitative levels ofGST or glutathione peroxidase gene expression in primary NBECs relativeto primary bronchogenic carcinoma tissue.

[0009] The inventors herein have discovered that interindividualvariation in GST enzyme gene expression translates into variation inrisk for bronchogenic carcinoma. For example, in some epidemiologicalstudies, GSTM1 null individuals have an increased risk (30, 31).However, the results of other studies are contradictory (32). Onehypothesis to explain these different results is that because themultiple GST and glutathione peroxidase enzymes have a broad substrateoverlap, a decrease in the expression level of one GST or glutathioneperoxidase may be compensated for by increased expression of another.Thus, the expression patterns for multiple relevant GST and glutathioneperoxidase enzymes may be more closely associated with risk than theexpression of each individual gene. Consequently, studies that do notcontrol for expression of all relevant genes may generate data that aredifficult to interpret.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a method to measure expressionof multiple target genes in a progenitor cell for bronchogenic carcinomausing reverse transcription-polymerase chain reaction (RT-PCR) to allowsimultaneous expression measurement of multiple target genes. Thequantitative competitive RT-PCR is used to measure mRNA levels ofglutathione-S-transferase (GSTs) and glutathione peroxidases (GSHPxs) inthe progenitor cell.

[0011] In a preferred method, at least one of the mRNA levels of thefollowing are measured: mGST, GSTM3, combined GSTM1, 2, 4, 5, GSTT1,GSTP1, GSHPx, and GSHPxA.

[0012] The levels of GSTP1, GSTM3 and GSHPx are significantly lower innormal bronchial epithelial cell than in bronchogenic carcinoma cells.In a preferred aspect, the gene expression index is evaluated bymultiplying the values for: MGST×GSTM3×GSHPx×GSHPxA×GSTP1. Thesensitivity for detecting normal bronchial epithelial cells as comparedto bronchogenic carcinoma cells is about 90% and the specificity fordetecting normal bronchial epithelial cells as compared to bronchogeniccarcinoma cells is about 76%.

[0013] In another aspect, the method comprises a) coamplifying ahousekeeping gene along with the target genes (to control for the amountof cDNA included in the reaction); b) including known amounts of cDNAcompetitive templates (CTs) for both the target genes and thehousekeeping gene (to control for the loss of predictable exponentialamplification with increasing cycles); c) identifying, choosing primersfor synthesizing the competitive templates (CTs) and for amplificationof native template (NT) and CT sequences; d) comparing the levels of thehousekeeping gene CTs to the target gene CTs where the ratio tohousekeeping gene CT to each of the target gene CTs is the same; e)preparing a master mix (sufficient for the PCR reactions) that containsthe components: dNTPs, buffer, water, Taq polymerase, cDNA and aliquotof CT solution containing known concentrations of CTs for thehousekeeping gene and the target genes; f) specifying each gene to beamplified in each reaction by the primers included in each reaction byaliquoting separately from the master mix; g) determining the amount ofcDNA loaded for each sample by comparing the density of PCR product bandfor housekeeping gene NT cDNA to PCR product band for housekeeping geneCT cDNA; and h) determining quantitative expression of the target genes.

[0014] The quantitative expression of the target genes is determined by:a) calculating a ratio of target gene NT to CT product; and b) dividingthe calculated number of target gene NT molecules by the calculatednumber of housekeeping gene NT molecules to correct for loadingdifferences.

[0015] The method of the present invention is especially useful fordetermining a patient who is at risk for developing cancer by assessingperipheral blood lymphocyte DNA for polymorphisms in a regulatory regionof target genes that are associated with high or low expression of thetarget genes.

DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 contains Table 1 which shows the demographic data ofindividuals without lung cancer and individuals with cancer.

[0017]FIG. 2 contains Table 2 which shows the primer sequences andproduct lengths of both native template (NT) and competitive template(CT) PCR products.

[0018]FIG. 3 contains Table 3 which shows the GST and peroxidase geneexpression (mRna/103 β-actin mRna) in primary bronchial epithelial cellsfrom subjects without bronchogenic carcinoma.

[0019]FIG. 4 contains Table 4 which shows the GST and peroxidase geneexpression (mRNA/103 μl-actin mRNA) in primary bronchial epithelialcells from subjects with bronchogenic carcinoma.

[0020]FIG. 5 contains Table 5 which shows the GST and peroxidase geneexpression (MRNA/103 β-actin mRNA) in cultured bronchial epithelialcells from subjects without bronchogenic carcinoma.

[0021]FIG. 6 contains Table 6 which shows gene expression test toidentify NBECs from subjects with bronchogenic carcinoma.

[0022]FIG. 7 shows representative agarose gels.

[0023]FIG. 8 shows glutathione peroxidase (A) or index values (B-E) forNBEC samples.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024] For many years, gene expression has been measured throughquantification of RNA by Northern or dot blot analysis. These techniquesrequire the amount of RNA obtainable from at least 1 cells for eachmeasurement. Often, a biopsy will provide only the number of cellsnecessary for a histological diagnosis and this is often far less than10⁵ cells. Recently developed PCR techniques allow measurement of RNAlevels in as few as 100 cells. However, techniques described thus farallow only qualitative, not quantitative measurement.

[0025] The present invention uses the using multiplex competitivereverse-transcriptase polymerase chain reaction amplification tosimplify and improve quantitative measurement of gene expression asdescribed in U.S. Pat. No. 5,876,978 to Willey et al. DNA extracted fromsamples is reverse transcribed and then subjected to PCR amplificationin the present of primers for both a “housekeeping” gene and targetsgene of interest.

[0026] The amount of a target DNA sequence is quantified within anidentified region of a selected cDNA molecule that is present within aheterogeneous mixture of cDNA molecules. It is to be understood thatmore than one targeted gene and/or housekeeping gene can be utilized andfurther that quantitation of such additional target and/or housekeepinggenes will necessitate the further inclusion of an internal standardcompetitive template comprising a mutation of that additional targetand/or housekeeping gene. It is to be understood that the mutatedcompetitive templates comprise at least one nucleotide that is mutatedrelative to the corresponding nucleotide of the target sequence. It isto be noted that mutation of only a single nucleotide that iscomplementary to the corresponding nucleotide of the housekeeping genesequence is required for the successful practice of the presentinvention. However, it is understood that longer deletions, insertionsor alternations are useful in the present invention. The target geneprimers (which serve as primers for both the native and competitivetemplates of the target gene), housekeeping gene primers (which serve asprimers for both the native and competitive template of the housekeepinggene), competitive templates of the target genes, and competitivetemplate of the housekeeping gene are subject to a PCR process alongwith native cDNA which contains the DNA for both the target genes andthe housekeeping gene. The PCR process provides cDNA products of 1)native cDNA of the target genes and the housekeeping gene and 2) mutatedcompetitive templates cDNA of the target genes and the housekeepinggene. The cDNA products are isolated using methods suitable forisolating cDNA products. The relative presence of the native cDNAproducts and the mutated cDNA products are detected by measuring theamounts of native cDNA coding for the target gene and mutated coding forthe competitive template of the target gene as compared to the amountsof native cDNA coding for the housekeeping gene and mutated cDNA codingfor competitive template of the housekeeping gene.

[0027] The terms primers, nucleic acids and oligonucleotides areunderstood to refer to polyribonucleotides and polydeoxyribonucleotidesand there is no intended distinction in the length of sequences referredto by these terms. Rather, these terms refer to the primary structure ofthe molecule. The terms include double and single stranded RNA anddouble and single stranded DNA. It is to be understood that theoligonucleotides can be derived from any existing or natural sequenceand generated in any manner. It is further understood that theoligonucleotides can be generated from chemical synthesis, reversetranscription, DNA replication and a combination of these generatingmethods. The term “primer” generally refers to an oligonucleotidecapable of acting as a point of initiation of synthesis along acomplementary strand when conditions are suitable for synthesis of aprimer extension product. The synthesizing conditions include thepresence of four different deoxyribonucleotide triphosphates and atleast one polymerization-inducing agent such as reverse transcriptase orDNA polymerase. These are present in suitable a buffer which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. It is understood thatwhile a primer is preferably a single strand sequence, such thatamplification efficiency is optimized, other double stranded sequencescan be practiced with the present invention.

[0028] The terms “target gene”, “sequence” or “target nucleic acidsequence” are meant to refer to a region of an oligonucleotide which iseither to be amplified and/or detected. It is to be understood that thetarget sequence resides between the primer sequences used to theamplification process.

[0029] The quantitative gene expression is measured by multiplexcompetitive PCR amplification of a) cDNA from at least one target geneof interest an at least one “housekeeping” gene and b) internal mutatedstandard competitive templates comprising base mutants of the targetgene of interest and the “housekeeping” gene cDNA that causes either aloss or gain of a restriction endonuclease recognition site. The methodcomprises the PCR amplification of a) cDNA from at least one target geneof interest and at least one “housekeeping” gene and b) competitivetemplates comprising sequences of the target gene of interest and the“housekeeping” gene that have been artificially shortened. Theseshortened sequences retain sequences homologous to both the target geneand the housekeeping gene primers used in PCR amplification. RNAextracted from sample cells or tissues are reverse transcribed. Serialdilutions of cDNA are PCR amplified in the presence of oligonucleotideshomologous to the target gene and the “housekeeping” gene, andquantified amounts of internal mutated standard competitive templates.The amplified DNA is restriction digested and electrophorsed, separatingnative from mutated products. Densitometry is performed to quantify thebands. This technique to measure the relative expression of a targetgene to a “housekeeping” gene is precise and reproducible for studiesdone with the same master mixture and dilution of internal standards.

[0030] Synthesized oligonucleotides homologous to any sequencescontaining a known restriction endonuclease recognition site or anysequence containing one or two-base pair mismatch for a knownrestriction endonuclease site that is present in the housekeeping genecan be utilized. The application of these restriction endonucleaserecognition sites is to either mutate the naturally occurring sites tonon-recognition sites or to mutate the mismatch sites to match sites, ineither case creating mutant sequences suitable for internal mutatedstandards competitive templates. The particular sites in thehousekeeping gene used for analysis of any particular other gene dependson the match and mismatch sites that are present in the other gene. Onedeterminant is the size of the DNA fragments that are generated from thehousekeeping gene and the target gene. It is desired that thesefragments separate well on gel electrophoresis.

[0031] Further, all oligonucleotides that contain sequences homologousto sequences in the genes for the housekeeping genes can be used in thepresent invention. Such homologous sequences may be used to generateartificially shortened competitive templates to the housekeeping genesgenerated according to the method described in the Willey et al. U.S.Pat. No. 5,576,978.

[0032] To identify and match one or two base mismatch sequences for allknown recognition sites, it is possible to use the Map program withinthe Genetics Computer Group software package (Devereux et al., supra.1984). The cDNA sequences are obtained for each gene, then each gene isevaluated for the presence of the match of one or two base pair mismatchsequences for every known restriction endonuclease. It is possible touse every gene containing any of these recognition sequences or one ortwo base pair mismatches of these sequences.

[0033] Multiplex competitive PCR improves and simplifies quantitation ofgene expression. Gene expression can be quantitated in very smallsamples of tissue or cells without resorting to radio labeling. As aresult, multiplex reverse transcription PCR is less expensive and saferto use than radio labeling. The results are reproducible for examplesusing the same master mixture and dilutions of internal mutated standardcompetitive templates.

[0034] It is to be understood that all oligonucleotides homologous toeach strand of the cDNA of known or potential housekeeping genes(including but not restricted to the human, mouse and rat GAPDH,β-actin, 28S RNA, 18S RNA, and all ribonucleic protein genes) andcontaining restrictions endonuclease recognition sites sequences or oneor two base pair mismatches for restriction endonuclease recognitionsequences are useful in the practice of the present invention. Theoligonucleotides are used to prepare competitive templates ofhousekeeping genes for use in quantitative PCR.

[0035] It is to be further understood that according to the method ofthe present invention, all oligonucleotides that contain sequenceshomologous to sequences in known or potential housekeeping genes(including but not restricted to GAPDH, β-actin, 28S RNA, 18S RNA, andall ribonucleic protein genes) are useful in generating artificiallyshortened competitive templates. The oligonucleotides are used toprepare competitive templates of housekeeping genes for use in thepresent invention.

[0036] It is contemplated that uses of this inventive technique include:a) evaluating gene expression from tissues obtained by endoscopic biopsy(brush or forceps), needle aspiration, and bone marrow biopsy; b)quantification of reporter gene expression in transient transfectionassays; and c) quantification of transfected genes following genetherapy.

[0037] It should be further understood that according to the method ofthe present invention, more than one gene can be evaluated at the sametime to determine the interindividual variation in antioxidant geneexpression that results in interindividual variation in risk forbronchogenic carcinoma.

[0038] Three genes, GSTM3, GSHPx, and GSTP1, are expressed at lowerlevels in NBECs from lung cancer patients compared to NBECs fromindividuals without lung cancer. Because GSHPx and GSTM3 each haveperoxidase activity, cells expressing low levels of these genes are moresusceptible to oxidant damage and carcinogenic transformation. Further,GSTM3 and GSTP1 metabolically inactivate PAH diol-epoxide carcinogens inNBECs; thus, decreased expression levels in NBECs lead to a decrease inthe cellular capacity to detoxify these carcinogens. It has beenreported that decreased expression of mouse GSTπ may be responsible forthe increased carcinogenicity of the PAH benzo(a)pyrene (41). GSTP1 wasexpressed at a higher level in NBECs from non-lung cancer patients thanthe other genes studied-herein. Recently described polymorphisms in thecoding region of GSTP1 have a strong association with increased risk forneoplasia (42, 43) and are important to assess along with GSTP1 geneexpression levels.

[0039] Although ˜50% of Caucasians lack GSTM1 expression due to a nullallele, NBECs from all 34 patients in this study expressed one or moreof these GSTM isoforms (See FIGS. 3 and 4). Because all of the GSTMisoforms have substrate overlap, it is possible that risk forbronchogenic carcinoma is not related to GSTM1 expression alone but alsoto relative gene expression levels of all GSTM isoforms in NBECs.

[0040] Non-cancer subjects 21 and 54 had mGST levels three logs and10-fold greater, respectively, than any of the other subjects. Such widefluctuation in gene expression was not observed for any of the othergenes. It is possible that a small segment of the population is capableof expressing very high levels of mGST either constitutively or uponexposure to certain xenobiotics. Because mGST has peroxidase activity(21) and because it was expressed at lower levels in the NBECs of lungcancer patients in this study (Tables 3 and 4), it would be expectedthat such a high level of expression would protect the cellular DNA fromoxidant damage and therefore lower cancer risk. The reason that mGSTexpression is not significantly different in the two groups, althoughthere is a 5-fold difference in the means, is that the subject 54 valueconfers such a high SD. If both subjects 21 and 54 are excluded fromanalysis, mean mGST expression is significantly lower (P<0.05) in thesamples from cancer patients.

[0041] Although protein and/or enzyme levels were not measured, mRNAlevels and enzyme activities for some of the measured genes and otherxenobiotic metabolism enzyme genes are known to be closely related. Forexample, Mosco et al. (44) reported in 1988 that GSTP1 enzyme activityand mRNA levels are highly correlated in several human breast cancercell lines. We have reported previously that CYP1A1 and NADPHoxidoreductase activities are correlated with mRNA levels inlymphoblastoid cell lines (35). CYP1A1 mRNA and enzyme activities alsohave been correlated in rat liver tissue (45). Further, manganesesuperoxide dismutase activity correlates with protein and mRNA levels infibroblasts (46).

[0042] Gene Expression Indices Identifies Individuals at Risk forBronchogenic Carcinoma.

[0043] An important feature of the method of the present invention isthat it allows expression values of multiple different genes to becombined into indices. Such index values are used to rank cell or tissuesamples. The gene expression indices generally correlate better thanexpression of any single gene or isozyme and phenotype. For the bestindex identified (mGST×GSTM3×GSHPx×GSHPxA×GSTP1) at a value thatprovided a sensitivity of ≧90%, the specificity was 76% (Table 6).Because 5-10% of smokers get lung cancer, it is reasonable tohypothesize that at least 5-10% of the people in the general populationhave a genetic predisposition to bronchogenic carcinoma. Thus, of thefour individuals without bronchogenic carcinoma who had index valuesbelow the cutoff value, one to two of them could be expected to be athigh risk for bronchogenic carcinoma if they smoked.

[0044] The manner in which gene expression values are combined intoindices depends in part on the weight given each gene. Indices arecalculated by multiplying gene expression values together so that eachgene expression value included has equal weight. One assumption made isthat, at the mean level of expression measured in NBECs, each of thegenes studied contributes equally to protection of NBECs from oxidantand/or carcinogen damage. This assumption is supported by theexpectation that the optimal level of expression for the function ofeach gene would be selected for through evolution. By combining geneexpression values into indices in a previous study (36) of bronchialepithelial cells, it is now possible to identify a gene expression indexthat is highly correlated with bronchogenic carcinoma by empiricallycombining multiple cell cycle gene expression values. This method isuseful for combining individual gene expression values into indices tobetter define the mechanisms underlying cellular phenotype.

[0045] Environmental Exposures Aspect Antioxidant Gene Expression.

[0046] The observed interindividual variation in the expression of GSTand GSHPx enzyme genes in primary NBECs (Tables 3 and 4) may result fromseveral different factors, including variation in constitutive level ofgene expression, variation in the inducible level of gene expression andvariation in inhalational exposure to exogenous oxidants, andxenobiotics in the form of cigarette smoke, occupational, orenvironmental pollutants. Although no significant relationship betweenantioxidant gene expression and present smoking or amount of pastsmoking (in pack-years) was observed, it remains possible that theinterindividual variation in gene expression observed could be due tovariation in exposure to xenobiotics and/or oxidants from sources otherthan cigarette smoke.

[0047] Lower mean antioxidant gene expression and interindividualvariation in expression among the cultured cells support the theory thatthe variation observed among the primary NBECs is at least in part dueto environmental rather than hereditary causes. Further, it is possiblethat hereditary differences caused variation in inducible as well asconstitutive levels of the genes tested. Thus, the NBECs of cancerpatients may express lower levels of GSTM3, GSHPx, and GSTP1 due to theinheritance of particular polymorphisms in the regulatory regions ofthese genes or of the transcription factors that bind to them.

EXAMPLE Materials and Methods

[0048] Reagents. 10×PCR buffer [500 mM Tris (pH 8.3), 2.5 mg/μl BSA, 30mM MgCl2] was obtained from Idaho Technology, Inc. (Idaho Falls, Id.).Taq polymerase (5 units/μl), oligo dT primers, Rnasin (25 units/μl),pGEM size marker, and dNTPs were obtained from Promega (Madison, Wis.).Moloney murine leukemia virus reverse transcriptase (200 units/μl), 5×first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2,50 mM DTT], and RNase-free water were obtained from Life Technologies,Inc. (Gaithersburg, Md.), NuSieve and SeaKem LE agarose were obtainedfrom FMC BioProducts (Rockland, Me.). TriReagent was obtained frommolecular Research Center (Cincinnati, Ohio), Bronchial epithelial cellgrowth medium was obtained from Clonetics (San Diego, Calif.). Naturalhuman fibronectin and collagen (type 1 rat tail) were obtained fromCollaborative Biomedical Products (Bedford, Mass.). All other chemicalsand reagents were molecular biology grade.

[0049] Samples. Primary NBECs were obtained by bronchial brush biopsy aspreviously reported (34, 35). This group of individuals without lungcancer consisted of healthy volunteers from a university setting,individuals under going diagnostic bronchoscopy, and three organ donors.The lungs of the donors did not meet criteria for transplantation due toCOPD (subjects 54 and 62) or asthma (subject 55). Two of the subjects(57 and 71) had bronchoscopy at the time of thoracotomy for resection ofadenocarcinoma of the colon that had metastasized to the lung. Subjects59 and 63-66 had bronchoscopy due to persistent hemoptysis or change incharacter of chronic cough, and no endobronchial mucosal lesions wereobserved. Samples from lung cancer patients were obtained viabronchoscopic bronchial brushing at the time of surgery as previouslyreported (36) or brushing of surgically resected samples (subjects 74and 75; Table 1). Samples that were evaluated in previous studies (34,35) have the same subject numbers in this study. Samples acquired sincethe time of those publications are numbered in order of acquisition.Cells were recovered from the bronchial brush into ice-cold 0.9% NaClsolution and pelleted. Informed consent was obtained from each patient.Demographic data are presented in Table 1.

[0050] RNA Extraction and Reverse Transcription. Excess NaClsolution/media was removed, and the cells were lysed in TriReagent.Total RNA was extracted according to the TriReagent ManufacturerProtocol (37). Following extraction, mRNAs were reverse-transcribedusing M-MLV reverse transcriptase and an oligo dT primer as previouslyreported (34).

[0051] Quantitative RT-PCR. Gene expression was determined usingquantitative competitive RT-PCR (33-35, 38). PCR reactions were cycled35 times in a Rapidcycler (Idaho Technology, Idaho Falls, Id.) in thepresence of two types of controls. First, a housekeeping gene (β-actin)was coamplified along with the target genes to control for the amount ofcDNA included in the reaction. Second, known amounts of cDNA CTs wereincluded for both the target and the housekeeping gene to control forthe loss of predictable exponential amplification with increasing cycles(38, 39). In these experiments, the concentration of the CTs in each PCRreaction was 10⁻¹⁴ M for β-actin and varied for each of the other genes.CTs were synthesized according to previously described methods (33, 40).Primers for synthesizing CTs and for amplification of NT and CTsequences were chosen using Oligo software (National Biosciences, Inc.,Plymouth, Minn.). After careful assessment of the sequences, we were notable to identify primers what would amplify GSTM1 without amplifyingGSTM2, 4, 5. Therefore, cDNA from all four isogenes were amplified withthe same primers. Sequences for mGST (GenBank accession no.J03746)(Forward: Seq. ID No. 22; Reverse: Seq. ID No. 23; CT: Seq. IDNo. 24), GSTM3 (J05459)(Forward: Seq. ID No. 13; Reverse: Seq. ID No.14; CT: Seq. ID No. 15), GSTM1, 2, 4, 5 (J03817, M63509, M96234,L02321)(Forward: Seq. ID No. 10; Reverse: Seq. ID No. 11; CT: Seq. IDNo. 12), GSTT1 (X79389)(Forward: Seq. ID No. 19; Reverse: Seq. ID No.20; CT: Seq. ID No. 21), GSHPx (Y00433)(Forward: Seq. ID No. 4; Reverse:Seq. ID No. 5; CT: Seq. ID No. 6), GSHPxA (D00632)(Forward: Seq. ID No.7; Reverse: Seq. ID No. 8; CT: Seq. ID No. 9), and GSTP1(X06547)(Forward: Seq. ID No. 16; Reverse: Seq. ID No. 17; CT: Seq. IDNo. 18) were retrieved from GenBank. Table 2 lists primer sequences andproduct lengths for both NT and CT PCR products. Primers for β-actin(Forward: Seq. ID No. 1; Reverse: Seq. ID No. 2; CT: Seq. ID No. 3) havebeen reported previously (34).

[0052] In each experiment, all of the genes were evaluated in a singlesample. Three stock mixtures of CTs were prepared, and these same threestocks were used for each experiment. Levels of housekeeping and targetgene CTs in the separate CT mixes were compared to each other. Thus, theratio of the housekeeping gene CT to each of the target gene CTs was thesame when each sample was assessed. For each experiment, a master mixsufficient for the planned number of PCR reactions was prepared thatcontained every component except the primers. The components of themaster mix were dNTPs, buffer, water, Taq polymerase, cDNA, and analiquot of CT solution. The CT solution contained known concentrationsof CTs for the housekeeping gene and the target genes. The gene to beamplified in each reaction was specified by the primers included in eachreaction tube, aliquoted separate from the master mix. Wheneverpossible, triplicate experiments were performed.

[0053] The amount of cDNA loaded for each sample was determined bycomparing the density of the PCR product band for β-actin CT cDNA.Quantification of expression of the target genes was determined in thefollowing way. First, the ratio of target gene nativetemplate:competitive template (NT:CT) product was calculated. Becausethe starting target gene CT concentration was known and the relativesimplification efficiencies for the NT and CT cDNAs were known (seebelow), the starting target gene NT cDNA concentration could bedetermined. Second, the calculated number of target gene NT moleculeswas divided by the calculated number of β-actin NT molecules to correctfor loading differences. Gene expression values are reported in Tables3, 4 and 5.

[0054] Visualization and Quantification. After amplification, productswere electrophoresed on 4% agarose gels (3:1 NuSieve:SeaKem) containing0.5 μg/ml ethidium bromide. Gels were visualized with a Foto/Eclipseimage analysis system (Fotodyne, Hartland, Wis.), and digital imageswere saved on a Power Mac 7100/66 computer as previously described (34).Collage software (Fotodyne) was used for densitometric analysis. Arepresentative gel is seen in FIG. 7.

[0055] Statistical Analysis. A Student's t test was used to investigatestatistical differences between primary NBECs from non-lung cancerpatients and primary NBECs from lung cancer patients for each gene orgene expression index. The Student's t test also was used to assess forsignificant differences in the expression value of any gene or geneexpression index due to gender. A two-factor ANOVA followed by aDuncan's Multiple Range Test was used to determine statisticallysignificant differences in gene expression levels and gene expressionindex values between present smokers, former smokers, and never smokers.A Pearson's correlation was used to determine whether there was astatistical association between the expression value for each gene orgene expression index with amount of past smoking (in pack-years). Therelationship between gene expression and age was assessed by the samemethod. All of Pearson's correlation tests were assessed for allindividuals, only lung cancer patients, only non-lung cancer patients,only present smokers, and only former smokers. X² analysis was conductedfor each gene or gene expression index using a range of cutoff values todetermine their sensitivity and specificity as a test for separatingcancer patients from non-lung cancer patients (Table 6). To determinestatically significant (P<0.05) interindividual variation in geneexpression levels among primary NBECs from non-lung cancer patients andprimary NBECs from lung cancer patients and compare this variation withinterindividual variation in cultured MBECs, a one-factor ANOVA wasused. All statistical analyses were conduced using SAS version 6.11 (SASInstitute, Cary, N.C.).

[0056] At least three replicate measurements were done for each geneexpression assessment when sufficient cDNA was available. Of the 280gene expression means reported in Tables 3, 4, and 5, three or morereplicate measurements were made for 218, and an SD is provided. Of theremaining 62 gene expression means reported, duplicate measurements wereobtained for 48, and a range is provided. There was sufficient cDNA foronly one measurement for 14 gene expression values.

Results

[0057] Reproducibility. Among the gene expression measurements for whichthree or more replicate values were obtained, the SD was <50% of themeans for 149 of 218, <75% for 190 of 218, and <100% for 210 of 218,with the SD >100% of the mean for 8 of 218 expression measurements(Tables 3, 4, and 5). This is similar to the reproducibility observed inother gene expression studies using the same method (33).

Comparison of Primary NBECs from Individuals With or WithoutBronchogenic Carcinoma

[0058] Individual Gene Expression Values. GSTM3, GSTP1, and GSHPX wereexpressed at significantly lower levels (P=0.02, 0.01, and 0.01,respectively) in primary NBECs from bronchogenic carcinoma patientscompared to primary NBECs from individuals without bronchogeniccarcinoma (bold font in Table 4). Of these genes, GSHPx was theindividual gene with the best sensitivity (80% for a value of 70-90mRNA/103 β-actin mRNA; Table 6). However, a value that was >90%sensitive had poor specificity (FIG. 8A).

[0059] For the other four genes tested, no significant difference ingene expression between the two groups of primary NBECs was observed.

[0060] Gene Expression Indices. Indices comprising expression values ofmultiple genes were formed by multiplying expression values of differentcombinations of genes together. Rather than assessing every possiblecombination of genes, 25 indices comprising the 5 genes thatindividually demonstrate the greatest difference between groups (GSP1,GSHPxA, GSTM3, mGST, and GSHPx; Table 6) were assessed. Index valueswere reported as molecules/10³ molecules of β-actin and were calculatedas the following example: (GSTM3 molecules/10³ molecules ofβ-actin×GSTP1 molecules/10³ molecules of β-actin×mGST molecules/10³molecules of β-actin=GSTM3×GSTP1×mGST molecules/10⁹ molecules ofβ-actin/10⁶ molecules of β-actin=index molecules/10³ molecules ofβ-actin.

[0061] For two indices that each comprised three genes(GSTP1×GSHPx×GSTM3; GSTP1×mGST×GSHPx), it was possible to identifycutoff values with sensitivities ≧90% and specificities >70% (Table 6;FIG. 8, B and C). These indices both included GSTP1 and GSHPx and variedonly with respect to the third gene (either mGST or GSTM3). For an indexthat included all four of these genes (mGST×GSTM3×GSHPx×GSTP1), a rangeof cutoff values (3.2×10⁻⁵-3.5×10⁻⁵ molecules/10³ molecules of β-actin)had a sensitivity of 100%. However, the specificity of this index wasonly 62% (Table 6). Reducing the cutoff value to 2.0×10⁻⁵ molecules/10³molecules of β-actin decreased the sensitivity to 90% but did notimprove the specificity (FIG. 8D). In addition, for an index comprisingfive genes (mGST×GSTM3×GSHPx×GSHPxA×GSTP1), a range of cutoff values(3.0×10⁻⁹-1.0×10⁻⁸ molecules/10³ molecules of β-actin) had a sensitivityof 90% and a specificity of 76% (Table 6; FIG. 8E).

[0062] Correlation with Age, Gender, and Smoking Age. Pearson'scorrelation was used to test the relationship of age to the expressionof each gene and the level of each gene expression index. First, thetest was run on all patients. Only GSHPx was significantly associated(negatively correlated) with age (P=0.018). To avoid bias caused by therelatively low representation of older individuals in the non-lungcancer group (mean age among non-lung cancer and lung cancer patientswas 39 and 69 years, respectively), the test also was run separately onthe lung cancer patients and the non-lung cancer patients. There was nosignificant association within either the non-lung cancer or the lungcancer group between age and GSHPx. GSHPx gene expression also wasassessed separately on samples from individuals aged 45-65 years. Inthis group, the mean age among nine non-lung cancer and four lung cancerindividuals was 54 and 55 years, respectively. As with the entire group,the mean level of GSHPx expression among the cancer cases (35.9molecules/10³ molecules of β-actin) was significantly lower (P=0.01)than the mean GSHPx expression among non-lung cancer cases (122molecules/10³ molecules of β-actin).

[0063] Smoking History. A Pearson's correlation was used to assessrelationships between smoking history and gene expression. This test wasrun once on all patients, once on present and former smokers only, onceon present smokers only, and once on former smokers only. No correlationbetween expression of any gene or gene expression index studied herewith smoking history (in pack-years) was observed among patients of anygroup.

[0064] Gender. Among the primary NBECs from lung cancer and non-lungcancer patients combined, no differences in gene expression or any geneexpression index were found due to gender.

[0065] Interindividual Variation in Gene Expression

[0066] Primary NBECs. There was significant (P<0.05) interindividualvariation in primary NBEC expression of each of the genes (Tables 3 and4). The value of mGST in NBECs from subject 21 was excluded fromstatistical analysis because it was an outlier (Table 3). Interpretationof this result is included in the discussion.

[0067] Cultured NBECs. In an effort to test whether the interindividualvariation in expression observed in primary NBECs was based onhereditary differences or environmental factors, gene expression wasmeasured in cultured NBECs from eight different individuals with nohistory of lung cancer. All of the cultures were maintained under theexact same conditions. This allowed hereditary differences inconstitutive gene expression to predominate. In these eight differentNBEC cultures, the mean level of expression for each antioxidant genestudied was lower than that observed among primary NBEC samples. Inaddition, although significant interindividual variation among culturedNBECs was observed for GSHPx, GSTM3, and mGST, it was less than thatobserved in primary NBECs (Tables 3, 4, and 5). Further, there was nosignificant interindividual variation in the expression of GSTM1, 2, 4,5, GSTT1, GSHPxA, or GSTP1 among cultured NBECs (Table 5).

[0068] The present invention involves a dramatic improvement overpreviously described approaches for evaluating interindividual aerationin risk for damage to normal bronchial epthiothial cells.

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1 24 1 21 DNA Homo sapiens 1 gattcctatg tgggcgacga g 21 2 20 DNA Homosapiens 2 ccatctcttg ctcgaagtcc 20 3 40 DNA Homo sapiens 3 ccatctcttgctcgaagtcc gccagccagg tccagacgca 40 4 20 DNA Homo sapiens 4 cctggtggtgctcggcttcc 20 5 20 DNA Homo sapiens 5 caatggtctg gaagcggcgg 20 6 40 DNAHomo sapiens 6 caatggtctg gaagcggcgg accggagacc aggtgatgag 40 7 21 DNAHomo sapiens 7 gcagagccgg ggacaagaga a 21 8 21 DNA Homo sapiens 8ctgctctttc tctccattga c 21 9 42 DNA Homo sapiens 9 ctgctctttc tctccattgacgctcttcct gtagtgcatt ca 42 10 20 DNA Homo sapiens 10 gggacgctcctgattatgac 20 11 20 DNA Homo sapiens 11 gcaaaccatg gccgcttccc 20 12 40DNA Homo sapiens 12 gcaaaccatg gccgcttccc ttctccaaaa tgtccacacg 40 13 20DNA Homo sapiens 13 gtgcgagtcg tctatggttc 20 14 20 DNA Homo sapiens 14agttgtgtgc ggaaatccat 20 15 40 DNA Homo sapiens 15 agttgtgtgc ggaaatccattgctctgggt gatcttgttc 40 16 20 DNA Homo sapiens 16 tccgctgcaa atacatctcc20 17 20 DNA Homo sapiens 17 tgtttcccgt tgccattgat 20 18 40 DNA Homosapiens 18 tgtttcccgt tgccattgat taggacctca tggatcagca 40 19 20 DNA Homosapiens 19 gctctacctg gacctgctgt 20 20 20 DNA Homo sapiens 20 ggaacacagggaacatcacc 20 21 40 DNA Homo sapiens 21 ggaacacagg gaacatcacc tagagcaggatggccacact 40 22 26 DNA Homo sapiens 22 caaaattgaa aaaatggttg acctca 2623 26 DNA Homo sapiens 23 tctatttggc tggggaaggg gtgtca 26 24 52 DNA Homosapiens 24 tctatttggc tggggaaggg gtgtcagggg tcgggaccac tcaaggaata ca 52

I claim:
 1. A method to measure expression of multiple target genes in aprogenitor cell for bronchogenic carcinoma comprising: using reversetranscription-polymerase chain reaction (RT-PCR) to allow simultaneousexpression measurement of the multiple target genes.
 2. The method ofclaim 1, which quantitative competitive RT-PCR is used to measure mRNAlevels of glutathione-S-transferases (GSTs) and glutathione peroxidases(GSHPxs) in the progenitor cell.
 3. The method of claim 2, in which theprogenitor cell comprises a bronchial epithelial cell.
 4. The method ofclaim 3, in which at least one of the mRNA levels of the followingglutathione-S-transferases are measured: mGST, GSTM3, combined GSTM1, 2,4, 5, GSTT1, GSTP1, GSHPx, and GSHPxA.
 5. The method of claim 4, inwhich the levels of GSTP1, GSTM3 and GSHPx are significantly lower innormal bronchial epithelial cell than in bronchogenic carcinoma cells.6. The method of claim 4, in which a gene expression index is evaluatedby multiplying the values for: mGST×GSTM3×GSHPx×GSHPxA×GSTP1.
 7. Themethod of claim 4, in which a gene expression index is evaluated bymultiplying the values for: mGST×GSTM3×GSHPx×GSTP1.
 8. The method ofclaim 4, in which a gene expression index is evaluated by multiplyingthe values for: GSTP1×mGST×GSHPx.
 9. The method of claim 4, in which agene expression index is evaluated by multiplying the values for:GSTP1×GSHPx×GSTM3.
 10. The method of claim 4, in which a gene expressionindex is evaluated by multiplying the values for: mGST×GSTM3×GSHPx. 11.The method of claim 4, in which a gene expression index is evaluated bymultiplying the values for: GSTM3×GSHPx.
 12. The method of claim 4, inwhich a gene expression index is evaluated by multiplying the valuesfor: GSTM×GSTP1×mGST.
 13. The method of claim 6, in which sensitivityfor detecting normal bronchial epithelial cells as compared tobronchogenic carcinoma cells is about 90%.
 14. The method of claim 6, inwhich specificity for detecting normal bronchial epithelial cells ascompared to bronchogenic carcinoma cells is about 76%.
 15. The method ofclaim 1, comprising a) coamplifying a housekeeping gene along with thetarget genes (to control for the amount of cDNA included in thereaction); b) including known amounts of cDNA competitive templates(CTs) for both the target genes and the housekeeping gene; c)identifying, choosing primers for synthesizing the competitive templates(CTs) and for amplification of native template (NT) and CT sequences; d)comparing the levels of the housekeeping gene CTs to the target gene CTswhere the ratio of housekeeping gene CT to each of the target gene CTsis the same; e) preparing a master mix that contains the components:dNTPs, buffer, water, Taq polymerase, cDNA and aliquot of CT solutioncontaining known concentrations of CTs for the housekeeping gene and thetarget genes; f) specifying each gene to be amplified in each reactionby the primers included in each reaction by aliquoting separately fromthe master mix; g) determining the amount of cDNA loaded for each sampleby comparing the density of PCR product band for housekeeping geneNTcDNA to PCR product band for housekeeping gene CTcDNA; and h)determining quantitative expression of the target genes.
 16. The methodof claim 15, in which the quantitative expression of the target genes isdetermined by: a) calculating a ratio of target gene NT to CT product;and b) dividing the calculated number of target gene NT molecules by thecalculated number of housekeeping gene NT molecules to correct forloading differences.
 17. The method of claim 15, in which thehousekeeping gene comprises β-actin.
 18. The method of claim 17, inwhich the concentration of the competitive templates (CTs) in each PCRreaction is 10⁻¹⁴M for β-actin and varied for each of the other genes.19. A method for determining a patient who is at risk for developingcancer by assessing peripheral blood lymphocyte DNA for polymorphesismsin a regulatory region of target genes that are associated with high orlow expression of the target genes.
 20. The method of claim 19, in whichquantitative competitive RT-PCR is used to measure mRNA levels ofglutathione-S-transferase (GSTs) and glutathione peroxidases (GSHPxs) ina progenitor cell.